Chromosomal aberrations giving rise to fusion genes are observed for many different leukemias (Rabbitts, T. H.; Nature 1994; 372: 143-149). Such tumor-specific oncogenes would be promising targets for new therapeutic approaches with increased specificity, if these oncogenes were important for maintaining the leukemic phenotype. However, in contrast to the development of a leukemia, a central role for its persistence has only been established for a minority of leukemic fusion genes.
The mixed lineage leukaemia (MLL) gene located on chromosome 11q23 is involved in numerous chromosomal translocations associated with human leukemia (Ernst, P., et al.; Curr Opin Hematol 2002; 9:282-287). The most prevalent among those is the translocation t(4;11)(q21;q23), which fuses MLL gene with the AF4 gene located on chromosome 4q21 (Gu, Y., et al.; Cell 1992; 71:701-708.; McCabe, N. R., et al.; Proc Natl Acad Sci USA 1992; 89:11794-11798; Domer, P. H., et al.; Proc Natl Acad Sci USA 1993; 90:7884-7888.). This translocation is the hallmark of a high-risk acute lymphoblastic leukemia (ALL) with a particularly poor prognosis in infants (Pui, C. H., et al.; Lancet 2002; 359:1909-1915).
The wild-type MLL gene is a member of the trithorax family and encodes for a 431 kD protein, which is proteolytically processed into two fragments −300 and 180 kD heterodimerizing with each other (Nakamura, T., et al.; Mol Cell 2002; 10:1119-1128; Yokoyama, A., et al.; Blood 2002; 100:3710-3718; Hsieh, J. J., et al.; Cell 2003; 115:293-303; Hsieh, J. J., et al.; Mol Cell Biol 2003; 23:186-194.). The MLL protein has a complex structure that includes an AT-hook domain for DNA-binding, a MT domain showing homology to DNA methyltransferase (DMT) and methyl binding domain protein 1 (MBD1), a plant homeodomain (PHD) containing zinc fingers and a SET histone methyl transferase domain (Ernst, P., et al.; Curr Opin Hematol 2002; 9:282-287). MLL is involved in mechanisms controlling hox genes transcription (Ayton, P. M., and Cleary, M. L.; Oncogene 2001; 20:5695-5707.). Interestingly, the Hox genes Hoxa7 and Hoxa9 in combination with the homeotic gene Meis-1 are necessary for the transformation induced by several different MLL fusion genes. Such a crucial role has not yet been reported for MLL-AF4. Nevertheless, expression levels of all three homeotic genes are raised in primary t(4;11) ALL.
The AF4 gene encodes a serine/proline-rich protein containing nuclear localization signal and GTP-binding domain. It localizes to the nucleus (Li, Q., et al.; Blood 1998; 92:3841-3847) and is probably involved in transcriptional activation functions. Whereas the MLL knockout is embryonally lethal (Yu, B. D., et al.; Proc Natl Acad Sci USA 1998; 95:10632-10636), AF4-deficient mice exhibit imperfect T-cell development and modest alterations in B-cell development (Isnard, P., et al.; Blood 2000; 96:705-710).
Notably, the t(4;11) translocation generates two fusion genes, AF4-MLL and MLL-AF4. The significance of either fusion gene for leukemogenesis is not completely understood yet. AF4-MLL has recently been shown to interfere with ubiquitin-mediated AF4 degradation and to transform murine embryonic fibroblasts (Bunsen, A., et al.; Oncogene 2004; 23:6237-6249). Ectopic expression of MLL-AF4 in t(4;11)-negative leukemic cell lines, however, inhibits cell cycle progression and triggers apoptosis (Caslini, C., et al.; Leukemia 2004; 18:1064-1071). Paradoxically, 20% of all (4;11) ALL patients lack AF4-MLL either on the transcriptional or genomic level, whereas MLL-AF4 is always detectable despite its proapoptotic activities upon ectopic expression (Downing, J. R., et al.; Blood 1994; 83:330-335; Reichel, M., et al.; Oncogene 2001; 20:2900-2907). Interestingly, several studies suggest that MLL-AF4 fusion oncogene supports cell survival in the t(4;11) context. Cells with t(4;11) translocation survive extended serum starvation (Kersey, J. H., et al.; Leukemia 1998; 12:1561-1564) and are resistant to CD95-mediated apoptosis (Dörrie, J., et al.; Leukemia 1999; 13:1539-1547).
To define the role of this fusion oncogene in leukemogenesis more precisely, we applied RNA interference (RNAi) to inhibit MLL-AF4 expression in leukemic cells. RNAi is a cellular process resulting in enzymatic cleavage and breakdown of mRNA, guided by sequence-specific double-stranded small interfering RNAs (siRNAs) (Dykxhoom, D. M., et al.; Nat Rev Mol Cell Biol 2003; 4:457-467). Cell transfection with siRNAs results in the generation of a cytoplasmatically located ribonucleoprotein complex called RNA-induced silencing complex. Upon activation of this complex by discarding one of the siRNA strands (Khvorova, A., et al.; Cell 2003; 115:209-216; Schwarz, D. S., et al.; Cell 2003; 115:199-208), the remaining strand targets RISC to complementary RNA sequences leading to the endonucleolytic cleavage of the target RNA by the RISC component Ago-2 (Meister, G., et al.; Mol Cell 2004; 15:185-197; Rand, T. A., et al.; Proc Natl Acad Sci USA 2004; 101:14385-14389; Song, J. J., et al.; Science 2004; 305:1434-1437). Exogenously added synthetic siRNAs were shown to act as very potent and sequence-specific agents to silence gene expression (Elbashir, S. M., et al.; Nature 2001; 411:494-498), demonstrating the great potential not only for the analysis of gene function but also for gene-specific therapeutic approaches (Cheng, J. C., Moore, T. B., and Sakamoto, K. M.; Mol Genet Metab 2003; 80:121-128; Heidenreich, O. Curr Pharm Biotechnol 2004; 5:349-354).
In the present study, we used RNAi to specifically inhibit MLL-AF4 gene expression in t(4;11) cells. We demonstrate that depletion of the fusion transcript MLL-AF4 inhibits clonogenicity and proliferation, induces apoptosis in t(4;11)-positive leukemic cells and compromizes their engraftment in a SCID mouse xenotransplantation model.